Autophagy is an evolutionarily conserved degradative eukaryotic cell pathway that plays a role in multiple cellular processes. One important function is as a key component of the cellular immune response to invading microbes. Autophagy has been found to directly target and degrade multiple intracellular bacterial species. In this thesis, I identify and characterize two distinct regulatory mechanisms for this pathway involving the second messengers: diacylglycerol and cyclic adenosine monophosphate (cAMP). Salmonella enteric serovar Typhimurium (S. Typhimurium) is a Gram-negative bacterial species that has been shown to be intracellularly targeted for degradation by autophagy. While targeting of this species has been previously shown to involve ubiquitination, this pathway accounts for only half of targeted bacteria. Here I show that ubiquitin-independent autophagy of S. Typhimurium requires the lipid second messenger diacylglycerol. Diacylglycerol localization to the bacteria precedes autophagy and functions as a signal to recruit the delta isoform of protein kinase C (PKC) in order to promote the specific autophagy of tagged bacteria. Furthermore, I have found that the role of diacylglycerol and PKC delta is not limited to antibacterial autophagy but also functions in rapamycin-induced autophagy indicating a general role for these components in this process. Multiple bacterial species have been found to be targeted by autophagy and while some have developed strategies that allow them to avoid targeting, no bacterial factor has yet been identified that is able to inhibit the initiation of this process. Here I show that two bacterial species, Bacillus anthracis and Vibrio cholera inhibit autophagy through the elevation of intracellular cAMP and activation of protein kinase A. Using two different bacterial cAMP-elevating toxins, I show that multiple types of autophagy are inhibited in the presence of these toxins. This is indicative of a general inhibitory function for these toxins and identifies a novel bacterial defence strategy. This work characterizes both a novel regulatory signal for the induction of autophagy and identifies a novel bacterial tactic to inhibit this process. Together the data presented in this thesis provide novel insight into the regulation of autophagy and offer potential targets for modulation of this process.

Abstract The phospholipid PI(3,4,5)P3 works as a second messenger in PI3K signaling pathway. The PI3K signaling pathway is involved in insulin stimulated nitric oxide (NO) production in vascular endothelium, leading to vasodilation and increased blood flow. However, the production of NO also has been reported in neurons as a neurotransmitter and in nucleus tractus solitarii (NTS), NO plays a role in central cardiovascular regulation. Previously, microinjection of insulin into the NTS of rats produces prominent depressor and bradycardic and activates the PI3K downstream Akt. Therefore, to investigate the detail downstream signaling of insulin stimulated NO production in NTS, the effects of PI(3,4,5)P3 on NO production were determined in neuronal cell lines PC12 and GH3 and in NTS of SD rats. The GH3 and differentiated PC12 exposed to 10

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 96-103).

Thesis (M. Sc.)--University of Alberta, 2010. / Title from pdf file main screen (viewed on Jan. 27, 2010). A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science, Department of Physics, University of Alberta. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 98-106).

Thesis (Ph. D.)--University of Missouri--Columbia, 2003. / "May 2003." Typescript. Vita. Includes bibliographical references (leaves 152-162).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 133-146).

Thesis (Ph. D.)--University of Nevada, Reno, 1998. / Includes bibliographical references. Online version available on the World Wide Web.

Human pluripotent stem cells (hPSCs) hold the potential to revolutionize cardiac tissue engineering. Because of their ability to proliferate and differentiate into all cardiomyocyte subtypes they represent an opportunity to regenerate virtually any tissue lost from the over 1 million cardiac disease patients in the United States alone. Studies have shown, however, that hPSCs which are not terminally differentiated pose a variety of risks including teratoma formation and lack of appropriate cell engraftment. It is therefore important to ensure that only well characterized cardiac subtypes are implanted into patients or used for research purposes. Current differentiation protocols generate a mixture of cardiac subtypes, and research on cardiac subtype specification is hampered by the lack of a high throughput method to distinguish cardiac subtypes. This thesis establishes the ability to identify, enrich and characterize cardiac subtypes using MBs. This will provide a robust tool for clinical use of hPSCs in cardiac cell therapy and for analysis of differentiation protocol effects on cardiac subtype formation.

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